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Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses 1 , 2 . Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood 3 , 4 . Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies. Scheiermann and colleagues show that circadian clocks control the infiltration of dendritic cells into skin lymphatics in mice and humans, with a peak migration to the lymph nodes during the rest phase.

Tumor cells frequently disseminate through the lymphatic system during metastatic spread of breast cancer and many other types of cancer. Yet it is not clear how tumor cells make their way into the lymphatic system and how they choose between lymphatic and blood vessels for migration. Here we report that mammary tumor cells undergoing epithelial–mesenchymal transition (EMT) in response to transforming growth factor-β (TGF-β1) become activated for targeted migration through the lymphatic system, similar to dendritic cells (DCs) during inflammation. EMT cells preferentially migrated toward lymphatic vessels compared with blood vessels, both in vivo and in 3D cultures. A mechanism of this targeted migration was traced to the capacity of TGF-β1 to promote CCR7/CCL21-mediated crosstalk between tumor cells and lymphatic endothelial cells. On one hand, TGF-β1 promoted CCR7 expression in EMT cells through p38 MAP kinase-mediated activation of the JunB transcription factor. Blockade of CCR7, or treatment with a p38 MAP kinase inhibitor, reduced lymphatic dissemination of EMT cells in syngeneic mice. On the other hand, TGF-β1 promoted CCL21 expression in lymphatic endothelial cells. CCL21 acted in a paracrine fashion to mediate chemotactic migration of EMT cells toward lymphatic endothelial cells. The results identify TGF-β1-induced EMT as a mechanism, which activates tumor cells for targeted, DC-like migration through the lymphatic system. Furthermore, it suggests that p38 MAP kinase inhibition may be a useful strategy to inhibit EMT and lymphogenic spread of tumor cells.

In rheumatoid arthritis (RA), synovial tissue abundantly expresses CCL21, a chemokine strongly associated with RA susceptibility. In this study, we aimed to characterize the functional significance of CCL21/CCR7 signaling in different phases of RA pathogenesis. We determined that CCR7 is a hallmark of RA M1 synovial fluid (SF) macrophages, and its expression in RA monocytes and in vitro differentiated macrophages is closely associated with disease activity score (DAS28). In early stages of RA, monocytes infiltrate the synovial tissue. However, blockade of SF CCL21 or CCR7 prevents RA SF-mediated monocyte migration. CCR7 expression in the newly migrated macrophages can be accentuated by LPS and IFNγ and suppressed by IL-4 treatment. We also uncovered that CCL21 stimulation increases the number of M1-polarized macrophages (CD14+CD86+), resulting in elevated transcription of IL-6 and IL-23. These CCL21-induced M1 cytokines differentiate naïve T cells to Th17 cells, without affecting Th1 cell polarization. In the erosive stages of disease, CCL21 potentiates RA osteoclastogenesis through M1-driven Th17 polarization. Disruption of this intricate crosstalk, by blocking IL-6, IL-23, or IL-17 function, impairs the osteoclastogenic capacity of CCL21. Consistent with our in vitro findings, we establish that arthritis mediated by CCL21 expands the joint inflammation to bone erosion by connecting the differentiation of M1 macrophages with Th17 cells. Disease progression is further exacerbated by CCL21-induced neovascularization. We conclude that CCL21 is an attractive novel target for RA therapy, as blockade of its function may abrogate erosive arthritis modulated by M1 macrophages and Th17 cell crosstalk.

Penile squamous cell carcinoma (PSCC) is an aggressive malignancy with a poor prognosis and limited therapeutic options. Tertiary lymphoid structures (TLSs) can support antitumour immunity, yet TLS maturation and maturation-associated stromal determinants in PSCC remain unclear. Here, we integrated hematoxylin and eosin (H&E) staining, multiplex immunohistochemical (mIHC) colocalization, spatial transcriptomics, single-cell RNA sequencing (scRNA-seq), and bulk RNA sequencing (bulk RNA-seq) to profile TLS maturity and TLS-associated stromal–immune features in PSCC. TLS maturity was quantified using an AUCell germinal centre-like signature, and the presence of mature TLSs (mTLSs) coincided with improved survival. We identified a CCL21 + CAF subset that was preferentially enriched in mTLSs, spatially concentrated in the mTLS-core and adjacent to B-cell-rich areas; mIHC further confirmed dense CCL21 + ACTA2 + CAFs near CD20 + aggregates. Along an inferred iTLS-to-mTLS continuum, the abundance of CCL21 + CAFs increased, and the expression of chemokines increased. Spatial and transcriptomic analyses further linked B-cell chemotaxis- and activation-related signatures to the CCL21-CCR7 axis, accompanied by germinal centre-like B-cell features. Clinically, higher CCL21 expression, elevated CCL21 + CAF signature scores, and stronger CCL21-CCR7 signatures in B cells were associated with favourable outcomes. Together, these data suggest that CCL21 + CAFs or CCL21 are potential prognostic biomarkers for risk stratification and immune microenvironment profiling and highlight the CCL21-CCR7 axis as a candidate pathway for therapeutic modulation of TLS maturity in PSCC. Graphical abstract

Humoral immune responses to immunization and infection and susceptibilities to antibody-mediated autoimmunity are generally lower in males 1 – 3 . However, the mechanisms underlying such sexual dimorphism are not well understood. Here we show that there are intrinsic differences between the B cells that produce germinal centres in male and female mice. We find that antigen-activated male B cells do not position themselves as efficiently as female B cells in the centre of follicles in secondary lymphoid organs, in which germinal centres normally develop. Moreover, GPR174—an X-chromosome-encoded G-protein-coupled receptor—suppresses the formation of germinal centres in male, but not female, mice. This effect is intrinsic to B cells, and correlates with the GPR174-enhanced positioning of B cells towards the T-cell–B-cell border of follicles, and the distraction of male, but not female, B cells from S1PR2-driven follicle-centre localization. Biochemical fractionation of conditioned media that induce B-cell migration in a GPR174-dependent manner identifies CCL21 as a GPR174 ligand. In response to CCL21, GPR174 triggers a calcium flux and preferentially induces the migration of male B cells; GPR174 also becomes associated with more Gαi protein in male than in female B cells. Male B cells from orchidectomized mice exhibit impaired GPR174-mediated migration to CCL21, and testosterone treatment rescues this defect. Female B cells from testosterone-treated mice exhibit male-like GPR174–Gαi association and GPR174-mediated migration. Deleting GPR174 from male B cells causes more efficient positioning towards the follicular centre, the formation of more germinal centres and an increased susceptibility to B-cell-dependent experimental autoimmune encephalomyelitis. By identifying GPR174 as a receptor for CCL21 and demonstrating its sex-dependent control of B-cell positioning and participation in germinal centres, we have revealed a mechanism by which B-cell physiology is fine-tuned to impart sexual dimorphism to humoral immunity. Male and female B cells show differing abilities to localize and contribute to germinal centres, in a way that depends on the G-protein-coupled guidance receptor GPR174 and its chemokine ligand CCL21.

PurposeGlioblastoma (GBM) is the most common and malignant primary adult brain tumor. Current care includes surgical resection, radiation, and chemotherapy. Recent clinical trials for GBM have demonstrated extended survival using interventions such as tumor vaccines or tumor-treating fields. However, prognosis generally remains poor, with expected survival of 20 months after randomization. Chemokine-based immunotherapy utilizing CCL21 locally recruits lymphocytes and dendritic cells to enhance host antitumor response. Here, we report a preliminary study utilizing CPZ-vault nanoparticles as a vehicle to package, protect, and steadily deliver therapy to optimize CCL21 therapy in a murine flank model of GBM.MethodsGL261 cells were subcutaneously injected into the left flank of eight-week-old female C57BL/6 mice. Mice were treated with intratumoral injections of either: (1) CCL21-packaged vault nanoparticles (CPZ-CCL21), (2) free recombinant CCL21 chemokine empty vault nanoparticles, (3) empty vault nanoparticles, or 4) PBS.ResultsThe results of this study showed that CCL21-packaged vault nanoparticle injections can decrease the tumor volume in vivo. Additionally, this study showed mice injected with CCL21-packaged vault nanoparticle had the smallest average tumor volume and remained the only treatment group with a negative percent change in tumor volume.ConclusionsThis preliminary study establishes vault nanoparticles as a feasible vehicle to increase drug delivery and immune response in a flank murine model of GBM. Future animal studies involving an intracranial orthotopic tumor model are required to fully evaluate the potential for CCL21-packaged vault nanoparticles as a strategy to bypass the blood brain barrier, enhance intracranial immune activity, and improve intracranial tumor control and survival.

Juvenile tissue healing is capable of extensive scarless healing that is distinct from the scar-forming process of the adult healing response. Although many growth factors can be found in the juvenile healing process, the molecular mechanisms of juvenile tissue healing are poorly understood. Here we show that juvenile mice deficient in the chemokine receptor CCR7 exhibit diminished large-scale healing potential, whereas CCR7-depleted adult mice undergo normal scar-forming healing similar to wild type mice. In addition, the CCR7 ligand CCL21 was transiently expressed around damaged cartilage in juvenile mice, whereas it is rarely expressed in adults. Notably, exogenous CCL21 administration to adults decreased scar-forming healing and enhanced hyaline-cartilage repair in rabbit osteochondral defects. Our data indicate that the CCL21/CCR7 axis may play a role in the molecular control mechanism of juvenile cartilage repair, raising the possibility that agents modulating the production of CCL21 in vivo can improve the quality of cartilage repair in adults. Such a strategy may prevent post-traumatic arthritis by mimicking the self-repair in juvenile individuals.

Hashimoto’s thyroiditis (HT) is the most common autoimmune disease characterized by lymphocytic infiltration and thyrocyte destruction. Dissection of the interaction between the thyroidal stromal microenvironment and the infiltrating immune cells might lead to a better understanding of HT pathogenesis. Here we show, using single-cell RNA-sequencing, that three thyroidal stromal cell subsets, ACKR1 + endothelial cells and CCL21 + myofibroblasts and CCL21 + fibroblasts, contribute to the thyroidal tissue microenvironment in HT. These cell types occupy distinct histological locations within the thyroid gland. Our experiments suggest that they might facilitate lymphocyte trafficking from the blood to thyroid tissues, and T cell zone CCL21 + fibroblasts may also promote the formation of tertiary lymphoid organs characteristic to HT. Our study also demonstrates the presence of inflammatory macrophages and dendritic cells expressing high levels of IL-1β in the thyroid, which may contribute to thyrocyte destruction in HT patients. Our findings thus provide a deeper insight into the cellular interactions that might prompt the pathogenesis of HT. Hashimoto’s Thyroiditis is an autoimmune disease with a complex pathomechanism. Authors here show by single cell RNA sequencing that the thyroidal microenvironment in the disease is characterised by three stromal cell subtypes that are potentially responsible for the recruitment of infiltrating inflammatory immune cells, such as macrophages and dendritic cells.

Chemotherapeutic drugs have been successfully used to treat several cancers, including melanoma. However, metastasis occasionally occurs after chemotherapy. Here, we reported that paclitaxel (PTX) treatment for B16F10 tumour in mice led to an enhanced lymphatic metastasis of the melanoma cells, although a significant inhibition of tumour growth at the injection site was observed. Further study demonstrated that PTX upregulated the expression of C-C chemokine receptor type 7 (CCR7) in B16F10 cells, enhancing their migration through the activation of JNK and p38 signalling pathways. Loss of CCR7 or blockade of C-C motif chemokine ligand 21 (CCL21)/CCR7 axis abolished the pro-migration effect of PTX on B16F10 melanoma cells. Importantly, combination of PTX and CCR7 mAb could simultaneously delay the tumour growth and reduce the lymphatic metastasis in B16F10 melanoma. The blockade of CCL21/CCR7 axis may collectively serve as a strategy for lymphatic metastasis in some melanoma after chemotherapy.